Ory 1001

The tested compounds have been dissolved in DMSO at 50 μM as stock concentration. All compounds have been tested at 10 μM and 50 μM as final concentrations for WB analysis. All compounds have been used at 0.1 μM, 5 μM, 10 μM, 25 μM, 50 μM as final concentration for proliferation assays. ORY-1001 (Selleck Catalog No. S7795) was used as reference compound at the concentration of 25 μM.

After stimulation for 48 h with the compounds at 10 μM and 50 μM, ORY-1001 (Selleck Catalog No. S7795), commercially available LSD1 inhibitor, was used as positive controls. ORY-1001 was used at the final concentration of 25 μM. Cells were collected and washed 2 times with PBS then processed for histone extraction. Pellets were resuspended in triton extraction buffer [TEB; PBS containing 0.5% Triton X 100 (v/v), 2 mmol/L PMSF, 0.02% (w/v) NaN₃], and the lysis was performed for 10 min at 4 °C. The samples were centrifuged at 2000× g for 10 min at 4 °C and pellets were washed in TEB (half volume). Samples were then resuspended in 0.2 N HCl, and acid histone extraction was carried out overnight at 4 °C. The supernatants were recovered, and protein concentration was quantified by Bradford assay (Bio-Rad). For each sample, 4 μg of proteins were loaded on 15% polyacrylamide gels. The nitrocellulose filters were stained with Ponceau red (Sigma-Aldrich, Schnellendorf, Germany) as an additional control for equal loading. H3K4me2, H3K9me2 (Diagenode, Ougrée, Belgium; pAB-035–050, pAb-060–050), and H4 (Cell Signalling #2592) were used according to the manufacturer’s instructions.

For AlphaLISA, 10000 cells/well were seeded on a 384-well plate (#6005350, Perkin Elmer, Waltham, MA, USA) using the MultiFlo FX Microplate Dispenser. The next day, cells were treated with LPS (L9143-02, Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 10 µg/mL, the plates were then incubated at 37 °C for 4 and 24 h, and the TNFa concentration was measured using AlphaLISA (Perkin Elmer, Waltham, MA, USA) on the Spark reader (Tecan, Männedorf, Switzerland) and the AlphaScreen module as per the manufacturer’s protocol. The CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) microplate assay was used to measure cell viability. Before the LPS challenge, cells were pre-treated for 1 h with the following drugs purchased from Selleckchem: AZD6244, SCH772984, MS-275, EX527, EPZ-5676, EPZ-6438, ORY-1001, RVX-208, OTX015, and (+)-JQ1 targeting the chromatin-associated proteins MEK 1/2, ERK 1/2, HDAC 1/2/3, SIRT1, DOT1L, EZH2, LSD1, BRD 2/3/4, BRD2/3, and BRD4 (Selleckchem, Houston, TX, USA), respectively, at the concentrations of 5, 1, 0.2, 0.04, 0.008, 0.0018, and 0.00032 µM. Drugs were dispensed using the Microlab STAR unit (Hamilton, Reno, NV, USA), while the transfer and media dilutions for the AlphaScreen readout were performed with the CyBio SELMA liquid handler (Analytic Jena, Jena, Germany). Measurements were performed in 12-plicate.